Mortality from lung cancer could be reduced through the identification of high-risk individuals and the implementation of chemopreventive strategies that can reverse or impede the progression of pre-malignant disease. Therefore, it is essential to develop biomarkers that can predict the efficacy of promising chemopreventive agents. In 1996, Dr. Larry Clark reported that the rate of expected lung cancer in a population taking selenium decreased by approximately 50%. This finding has led to the implementation of ECOG 5597, A Phase III Chemoprevention Trial of Selenium Supplementation in Persons with Resected Stage I Non-Small Cell Lung Cancer. This trial is testing the hypothesis that 200 5g of L-selenomethionine given as selenized yeast can decrease the rate of second primary tumors in patients who have undergone curative surgery for stages Ia (T1N0) or Ib (T2N0) non-small cell lung cancer. Our studies have identified genes inactivated by aberrant cytosine-guanosine (CpG) island methylation as candidate biomarkers for early detection of lung cancer. A nested, case-control study of high-risk smokers with chronic obstructive pulmonary disease revealed that a panel of genes could predict incident lung cancer 3 - 18 months prior to clinical diagnosis. Specifically, concomitant methylation of three or more of a six-gene panel was associated with a 6.5-fold increased risk and a sensitivity and specificity of 64%. Our studies during the initial funding period of this grant have used some of the more promising methylated genes identified in that study as biomarkers to evaluate methylation in the sputum and blood of persons participating in the Phase III selenium chemoprevention trial. Several key findings have emerged. First, through comparison of selenium trial participants to cancer-free smokers and never smokers, we demonstrated that the prevalence of gene methylation in plasma and sputum increased with lung cancer risk. These findings support gene methylation as a biomarker for assessing response to selenium and predicting cancer recurrence. Second, a preliminary analysis demonstrated a significant association between gene methylation in sputum collected at entrance onto the clinical trial and the recurrence of lung cancer. Third, through a supplement, we evaluated drug combinations for chemoprevention. Treatment of A/J mice 42 weeks after carcinogen exposure with rosiglitazone, a PPAR-3 agonist; sodium phenylbutyrate, a histone deacetylase inhibitor, hydralazine, a 5- methylcytosine demethylating agent, and selenium markedly decreased the progression and size of pre- invasive lesions in the A/J mouse lung. Work in this competitive renewal will determine the ability of a panel of genes inactivated through methylation in sputum or plasma to predict response to therapy, and the recurrence of cancer, and to define potential drug combinations to be evaluated for primary and secondary prevention in future studies. Project Narrative: These studies will determine whether genetic biomarkers that can be assessed in sputum and plasma can predict response to selenium, a natural compound being tested for the prevention of cancer in persons previously operated on for early stage lung cancer. Studies will also assess whether these same biomarkers can predict cancer recurrence. Finally, a cocktail of agents with minimal side effects is being evaluated that could ultimately be used in the prevention of lung cancer in humans.